Celltiter glo ctg 2.0 luminescent cell viability assay

A CellTiter-Glo (CTG) 2.0 Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to determine the effect of miR-22 and miR-205 on the cells’ viability. Briefly, 100 μL of cell suspension was dispensed in the Perkin Elmer ViewPlate-96 microplate with a clear flat bottom and black well walls (Perkin Elmer Inc., Waltham, MA, USA) for a final concentration of 1000 cells per well. After 72 hours, 100 μL of the CellTiter-Glo reagent was dispensed into the wells, and the luminescence reads were measured using a PHERAstar plate reader (BMG Labtech, Ortenberg, Germany).

Cell viability tests were performed to all cell lines cultured either on top of the Myogel or on top of plastic wells. For Myogel assay, the 96-well Transwell plate was coated with 50 μl Myogel (0.5 mg/ml) and left to settle overnight in the cell culture incubator. The excess of the coating was removed carefully with suction, and 1000 cells in 100 μl media per well were added. The cells were allowed to grow for 72 h at + 37 °C. CellTiter-Glo® (CTG) 2.0 Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to determine the cells viability. The plate was taken out from the incubator to room temperature for 30 min before starting the assay. One hundred microliters of CellTiter-Glo was dispensed in each well. The plate was put on a plate shaker (Heidolph, Schwabach, Germany) for 5 min at 450 rpm and then in plate spinner (Thermo Scientific, Massachusetts, USA) for 5 min at 1000 rpm. The plate was then placed in the BMG PHERAstar FS (BMG Labtech, Offenburg, Germany) plate reader to detect cell viability. The results represent an average of three independent experiments.

After 3 days, the CellTiter-Glo^® (CTG) 2.0 Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to determine the efficacy of the drugs. We first imaged the plates using IncuCyte Live-Cell Imaging System (Sartorius, Göttingen, Germany) to observe cell density (Figure S4). We then transferred the plates to room temperature for 15 min and dispensed CTG to the assay plates using the MultiFlo™ FX automated reagent dispenser (30 µL/well). The plates were shaken for 10 min at 450 rpm and then centrifuged for 5 min at 1000 rpm. Complete lysis of the cells was observed under a microscope. The CTG signal was detected using the PheraStar plate reader (BMG Labtech, Ortenberg, Germany).